Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 12, 2018 to March 16, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

High-performance liquid chromatography (HPLC) method

Details on sampling:

To demonstrate that test vessels were dosed with nominal exposure concentrations of test substance, whole test vessels were analysed for test substance using the liquid chromatography mass spectrometry method.

Vehicle:

yes

Remarks:

Tetrahydrofuran (THF)

Details on test solutions:

Preparation of test solutions
This study was run with a culture medium control, solvent control and nominal exposure concentrations of 0.011, 0.037, 0.117, 0.375 and 1.2 mg/L. A primary stock concentrate of test substance, with a nominal concentration of 12 g/L, was prepared by adding a nominal 0.120 g of test substance (actual weight 0.12004 g) and making up to 10 mL with the solvent Tetrahydrofuran (THF) in a volumetric flask. The stock was sonicated for 60 minutes and observed to be a slightly hazy, colourless solution. The primary stock was used to prepare an intermediate stock with a nominal concentration of 1.2 g/L by the addition of 1 mL of the primary stock to a volumetric flask and making up to 10 mL with THF. This intermediate stock was shaken to mix and observed to be a clear and colourless solution. The primary and intermediate stocks were used to prepare the test solutions by direct addition of the appropriate amount, using a microliter syringe, to stirring dilution water in a volumetric flask. The solvent control was prepared in the same way using solvent only. Solvent only additions were also made, as appropriate, such that the final concentration of solvent used in all exposure solutions was 0.1 mL/L with the exception of the dilution water control which contained dilution water only. All test solutions were assessed to be clear and colourless prior to use. The appropriate test solution (100 mL volume) was dispensed into each test and blank vessel.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 3-d old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium and under the environmental conditions described for the test. The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium.

Test type:

not specified

Water media type:

other: AAP-medium

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 ± 2ºC

pH:

7.09 to 7.46

Nominal and measured concentrations:

A top concentration of 1.2 mg/L was selected based on the solubility and toxicity observed in a non-GLP solubility trial and range-finding study. This concentration was dosed to the media via a solvent carrier (Tetrahydrofuran (THF)) to assist in dosing the test compound. The concentration of solvent used in all exposure solutions with the exception of the control was 100 µL/L.
Control, solvent control, 0.011, 0.037, 0.117, 0.375 and 1.2 mg/L (nominal)
Control, solvent control, 0.0051, 0.019, 0.063, 0.16 and 0.39 mg/L (mean measured)

Details on test conditions:

Appratus
The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 22 2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

> 0.39 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

ca. 0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.019 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 0.063 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

ca. 0.06 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EyC10

Effect conc.:

ca. 0.026 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

biomass

Results:

Analytical data

The limit of quantification (LOQ) of Test substance in this study was 0.0050 mg/L for all test solutions. The instrument LOQ was 0.0025 mg/L but during analysis, samples from the controls and each test concentration were diluted ×2, doubling the LOQ. All analytical values are quoted to two significant figures and percentages to the nearest integer. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.995. The maximum percentage difference from nominal concentration for standards at the LOQ was less than 20% and less than 15% at levels greater than the LOQ. On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

Biological data

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass.

Growth rates

The growth rates were examined by a two-sample t-test to identify significant differences (p <0.05) between the control and solvent control. There was no significant effect between the control and solvent control. The solvent control was therefore compared to the treatments by one-way analysis of variance and Bonferroni adjusted t-tests to identify significant differences (p <0.05). The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method.

The mean growth rates, are given in below table together with the rates expressed as percentages of the control.

Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Mean growth rate/day (0-72 h)	Mean growth rate/day 95% Cl	Percentage of solvent control (%)
Culture medium control	0	1.361	1.334-1.389	98
Solvent control	0	1.382	1.344-1.421	-
0.011	0.0051	1.386	1.386-1.328	100
0.037	0.019	1.37	1.320-1.419	99
0.117	0.063	1.235*	1.155-1.316	89
0.375	0.16	0.973*	0.586-1.360	70
1.2	0.39	0.709*	0.554-0.863	51

* Significant differences (p <0.05) from the control

All biological measurements are quoted to 3 decimal places and percentages to the nearest integer.

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)	95% confidence limits (mg/L)
NOEC	0.019	-
LOEC	0.063	-
ErC50	>0.39	-
ErC20	0.11	-
ErC10	0.060	-

Yield

This response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. These cell particle densities were examined by two-sample t-test to identify significant differences (p <0.05) between the control and solvent control. There was no significant effect between the control and solvent control. Therefore, the solvent control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t-tests to identify significant differences (p <0.05). The EC50, EC20 and EC10 values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method.

The yield mean values are shown in below table, together with the yields expressed as percentages of the control:

Nominal concentration of test substance (mg/L)	Mean measured concentration of test substance (mg/L)	Mean yield (104 Cells/mL) (72 h)	Percentage of solvent control (%)
Culture medium control	0	32.62	93
Solvent control	0	34.92	-
0.011	0.0051	35.21	101
0.037	0.019	33.47	96
0.117	0.063	22.21*	64
0.375	0.16	10.54*	30
1.2	0.39	5.95*	17

* Significant differences (p <0.05) from the control

Mean yield values are quoted to 2 significant figures and percentages to the nearest integer.

The results obtained from these statistical analysis, based on mean measured test concentrations, were as follows:

	Test substance (mg/L)	95% confidence limits (mg/L)
NOEC	0.019	-
LOEC	0.063	-
EyC50	0.10	-
EyC20	0.040	-
EyC10	0.026	-

Additional biological data

The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the solvent control and each test concentration appeared normal.

Validity criteria

The validity criteria specified in the OECD 201 guideline are;

1) To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 h test period. In this test, cell particle density increase (measured as a surrogate for biomass) were 59.5 and 63.6 over the 72 h for the control and solvent control respectively.

2) The mean coefficients of variation for control replicate sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 13% for control and 10% for solvent control..

3) The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and in this test, was calculated to be 1.9% for control and 2.7% for solvent control.

Based on the study results, it was concluded that the study has fulfilled validity criteria.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, 72 h ErC50, EyC50, ErC10, EyC10, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.39, 0.10, 0.06, 0.026, 0.019 and 0.063 mg/L, respectively

Executive summary:

A study was conducted to determine the acute toxicity study of the test substance, C16-18 AMP, to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and solvent control (Tetrahydrofuran) and triplicates of each concentration of the test substance were employed. The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 µm respectively. Each replicate test vessel was inoculated with 0.446 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.558 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0.011, 0.037, 0.117, 0.375 and 1.2 mg/L in AAP medium for 72 h. Analytical dose verification was conducted via HPLC. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0.0051, 0.019, 0.063, 0.16 and 0.39 mg/L. Therefore, the test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The ErC50 and ErC10 values, based on growth rate and the EyC50 and EyC10 values, based on cell particle density were calculated to be >0.39, 0.06, 0.10 and 0.026 mg/L respectively. The NOEC and the LOEC values were determined to be 0.019 mg/L and 0.063 mg/L, respectively, for both growth rate and cell particle densities. The study results were found to have fulfilled the all the Guideline required validity criteria. Under the study conditions, 72 h ErC50, EyC50, ErC10, EyC10, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.39, 0.10, 0.06, 0.026, 0.019 and 0.063 mg/L, respectively (Scymaris, 2018).​

Description of key information

The 72 h ErC50, EyC50, ErC10, EyC10, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.39, 0.10, 0.06, 0.026, 0.019 and 0.063 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.39 mg/L

EC10 or NOEC for freshwater algae:

0.06 mg/L

Additional information

A study was conducted to determine the acute toxicity study of the test substance, C16-18 AMP, to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and solvent control (Tetrahydrofuran) and triplicates of each concentration of the test substance were employed. The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 µm respectively. Each replicate test vessel was inoculated with 0.446 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.558 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0.011, 0.037, 0.117, 0.375 and 1.2 mg/L in AAP medium for 72 h. Analytical dose verification was conducted via HPLC. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0.0051, 0.019, 0.063, 0.16 and 0.39 mg/L. Therefore, the test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. The ErC50 and ErC10 values, based on growth rate and the EyC50 and EyC10 values, based on cell particle density were calculated to be >0.39, 0.06, 0.10 and 0.026 mg/L respectively. The NOEC and the LOEC values were determined to be 0.019 mg/L and 0.063 mg/L, respectively, for both growth rate and cell particle densities. The study results were found to have fulfilled the all the Guideline required validity criteria. Under the study conditions, 72 h ErC50, EyC50, ErC10, EyC10, NOEC and LOEC values for the test substance with freshwater green algae, were determined to be >0.39, 0.10, 0.06, 0.026, 0.019 and 0.063 mg/L, respectively (Scymaris, 2018).​